Determination of the intrinsic Michaelis constant of immobilized alpha-chymotrypsin.

The Michaelis constant of alpha-chymotrypsin, immobilized on a glutaraldehyde-activated silicate support, for N-glutaryl-L-phenylalanine-p-nitroanilide was determined and was found to be identical with that of the enzyme in solution. The influence of intraparticular diffusion was taken into account by immobilizing different amounts of enzyme, thus changing the magnitude of diffusional constraints and extrapolating apparent Michaelis constants, determined for each amount of immobilized enzyme, to zero diffusional constraints. The possible effect of the immobilized enzyme distribution inside the porous matrix was investigated through numerical simulations.